Epicypher：EpiDyne-FRET Nucleosome Remodeling Assay Substrate

時間：2023/12/14閱讀：515

當與酶活性重塑復合物如SMARCA4、SMARCA2和ACF（如EpiCypher貨號15-1014、15-1015、15-1013）配對時，該產品提供核小體重塑的高通量熒光讀數。EpiDyne®-FRET單核小體由在大腸桿菌中表達的重組人組蛋白（組蛋白H2A-Cy5、H2B、H3.2和H4各兩種；accession numbers:：H2A-P4908；H2B-O60814；H3.2-Q71DI3；H4-P62805）組裝而成，該組蛋白由212個DNA堿基對纏繞，并攜帶5'Cy3、Widom 601核小體定位元件和重塑時暴露的DpnII限制性內切酶基序（GATC）[1]。H2A在殘基120位處具有Thr-Cys取代，其中Cy5是共軛的。H3.2在殘基110位處具有Cys-to-Ala取代。

[1] Lowary & Widom J Mol. Biol. (1998) PMID: 9514715.

產品詳情

保存溫度:Stable for six (6) months at -80°C from date of receipt. For best results, aliquot and avoid multiple freeze/thaws.

運輸溫度:Dry ice.

產品形式:Purified recombinant mononucleosomes, (21.8 µg protein weight, 50 µg DNA+protein) in 44.2 µL of 10 mM Tris-HCl pH 7.5, 25 mM NaCl, 1 mM EDTA, 2 mM DTT, 20% glycerol. MW = 240,650 Da. Molarity = 4.7 µM.

驗證數據

	Figure 1: DNA Gel Data EpiDyne-FRET substrate resolved via native PAGE gel and stained with ethidium bromide to visualize DNA. Lane 1: Free DNA (100 ng). Lane 2: Intact EpiDyne-FRET nucleosomes (400 ng).
	Figure 2: Protein Gel Data Coomassie stained PAGE gel of proteins in EpiDyne-FRET substrate (1 µg) demonstrates the purity of histones in the preparation. Sizes of molecular weight markers and positions of the core histones (H2A-Cy5, H2B, H3.2, and H4) are indicated.

Figure 3: Nucleosome Remodeling Data ACF1/ATP-dependent nucleosome remodeling reaction. EpiDyne-FRET nucleosomes (15 nM) were incubated with ACF1 chromatin remodeler (EpiCypher 15-1013; indicated concentrations) in the presence or absence of 1 mM ATP. Upon the addition of ATP, reactions were immediately read in an Envision Multilabelplate reader (PerkinElmer). Data are presented as the mean of the Cy3/Cy5 ratio (N=2).